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Abstract:
Background: Purification of virus particles for vaccines and viral vectors for gene therapy is a major large-scale separations challenge. Here we consider the purification of human influenza virus as a model system for enveloped viruses of about 100 nm in diameter.
Methods: Virus produced in roller bottles (250 mL per bottle) and microcarrier cultures (2-3 L wv) was clarified by two static filtration steps and inactivated with ß-propiolactone. The use of tangential flow filtration and dialfiltration was investigated in order to concentrate the virus and to reduce the level of contaminating compounds. Small-scale hollow-fibre modules (40..200 cm2 filter area, Amersham Biosciences) with different molecular weight cut-offs and pore sizes (500 kDa, 750 kDa, 0.1 µm, 0.45 µm) were used in this study. Rejection of virus particles, permeation of protein and DNA, permeate flux and flux decline were examined for all the membranes.
Results: No virus was found in the permeates of the 500 kDa and 750 kDa membranes whereas complete breakthrough was achieved with the 0.45 µm membrane. Virus particles seemed to enter the pores of the 0.1 µm membrane leading to very low recoveries. Most of the contaminating proteins could be removed by diafiltration using a 750 kDa membrane. Only partial removal of DNA could be achieved independent of the membrane type.
Conclusion: 750 kDa hollow-fibre membranes seem to be an ideal choice for the concentration of influenza virus. They resulted in high recoveries of the product whereas most of the contaminating proteins could be removed. The partial removal of DNA suggests that genomic DNA fragments are either about the same size as or ar